Project Summary The long term goal of this research is to determine the mechanism and effect of dopamine signaling on hormone secretion from the islet of Langerhans. Blood glucose homeostasis depends on glucose-stimulated insulin secretion (GSIS) from islet 2 cells, but we also know that non-glucose signals are important for modulating insulin secretion, and many of these signals are known to communicate through G-protein coupled receptors. Dopamine acts through a number of G-protein coupled receptors, but its signaling in the 2-cell has not been well-characterized. All current antipsychotic drugs block D2 dopamine receptors as well as other targets, and the most effective second generation antipsychotics cause significant metabolic side-effects leading to increased risks of obesity and diabetes. Unfortunately, there is no consensus about the molecular mechanisms underlying the side-effects. Since all of these anti-psychotics block D2 receptors, but not all have notable metabolic side-effects, it has been assumed that the side-effects are mediated by interactions with molecules other than D2 receptors. The 2-cell has drawn little attention as a potential target of these drugs, presumably because there are no known dopaminergic neurons terminating in the islets, nor do sufficient levels of dopamine circulate through the blood stream. However, L-DOPA does circulate in the blood stream at appropriate levels, and 2-cells contain the molecular machinery needed to convert L-DOPA to dopamine. 2- cells also express the D2 receptor and VMAT-2, which loads dopamine into secretory granules. Thus, a link may exist between D2 receptor inhibition and 2-cell dysfunction, but investigations of this link will depend on a more complete understanding of dopamine action in the 2-cell. Towards this understanding, we hypothesize that 2-cells take up L-DOPA, synthesize dopamine and package it in the insulin secretory granules, and that stimulated secretion of dopamine acts through an autocrine loop to down-regulate subsequent insulin secretion. To test this hypothesis, we will utilize state-of-the-art live cell imaging methods, many of which have been pioneered by our laboratory. These novel techniques allow us to examine intracellular and multicellular events with unprecedented specificity and resolution. We have also developed several novel microfluidic devices for the study of pancreatic islets and their secreted products, and these can all be coupled with our unique quantitative imaging approaches to provide an ideal platform for many of the proposed experiments. In this proposal, we will refine and extend our established live cell imaging approaches to address the following four specific aims: 1) to determine the effects of L-DOPA on 2-cells in terms of the generation of dopamine and resulting modulation of the intracellular signaling pathways involved in GSIS; 2) to determine the intracellular signaling pathways and functional targets of dopamine receptor activation in the 2-cell; 3) to determine the role of expression and trafficking of the D2 receptor and the dopamine transporter in these signaling pathways; 4) to determine the effects of various second generation antipsychotic drugs on these pathways.